Rapid glycan labelling with fluorogenic molecular rotor dyes and nucleic acid stains responding to changes of the cell surface

Herein, we introduce a dual-labelling technique for covalent modification of cell surfaces with viscosity-sensitive and viscosity-insensitive fluorescent dyes. For this purpose, we combined an improved method for chemical reactions at sialic acid residues with fluorescent molecular rotor (FMR) dyes and a dye commonly used to stain nucleic acids. Fluorescence of FMR dyes increases with increasing viscosity providing options for detecting changes of cell surface viscosity during disease and therapeutic intervention. The method involves mild periodate oxidation of cell surface glycans followed by organocatalysed dye conjugation. Both steps are performed within a total of 15 min at 25 °C, avoiding cold stress of cells. We show that FMR dyes such as 9-(2-Carboxy-2-cyanovinyl)julolidine (CCVJ) or Cyanine 3 (Cy3) and the nucleic acid stain thiazole orange (TO) emit sufficiently strong signals to allow fluorescence microscopy imaging of surfaces of A549, HeLa and CEM cells. We demonstrate that the fluorescence of CCVJ and TO on cell surface glycans and isolated mucin is influenced by the action of the disulfide-cleaving agent TCEP. Co-labelling of cells with CCVJ and an environmentally insensitive AF568 dye revealed a TCEP-induced decrease in the intensity CCVJ fluorescence, which did not occur when the dye was studied in glycerol solution, consistent with a decrease of the fluorescence lifetime determined by fluorescence lifetime imaging microscopy (FLIM). This indicates that glycan-bound FMR dyes respond to changes in friction that hinder dye intramolecular rotation, potentially enabling the detection of viscosity variations in cell surface layers.